JP2005245302A - Recombinant influenza virus and vaccine using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective vaccine against H5N1 type influenza. <P>SOLUTION: A virus strain having low pathogenesis and high immunogenicity is prepared by modifying a hemagglutinin gene in order to reduce the number of a basic amino acid existing in rows on the cleavage site of a hemagglutinin protein of H5N1 type influenza. The vaccine is prepared by using the virus strain. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a recombinant influenza virus, more specifically, an attenuated influenza virus that has been genetically modified using a base sequence obtained by modifying the surface antigen gene of a highly virulent highly pathogenic H5N1 influenza virus, and It relates to an influenza vaccine using the same.

  Influenza is one of the serious viral infectious diseases that can be prevalent on a global scale, and the establishment of an effective prevention method is strongly desired. However, influenza viruses have a relatively high frequency of antigen mutations, and a vaccine that can sufficiently cope with influenza viruses having mutated antigens and can prevent influenza infection at a high probability has not yet been developed.

Influenza viruses are classified into subtypes such as A Soviet type (H1N1) and A Hong Kong type (H3N2) depending on the type of hemagglutinin (HA) protein or neuraminidase (NA) protein, which is a major surface antigen present on the surface of the virus particle. being classified. Influenza viruses such as subtype H5 and subtype H7 that use birds as a host are known as virulent types (Non-patent Documents 1 and 2). In addition, it has also been known that influenza viruses hosted by other species of animals such as birds directly infect humans (Non-patent Document 3). In fact, since May 1997, it was confirmed in birds until then. A number of infected people with subtype H5 influenza virus (H5N1), which have not been found in humans, have been confirmed, and it is required to produce vaccines against these influenza viruses as soon as possible.

Y. Kawaoka et al., Virology 158: 218-227 (1987) J.A.Walker and Y. Kawaoka, J.General Virol. 74: 311-314 (1993) K. Subbarao et al., Science 279: 393-396 (1998)

In order to produce a vaccine against influenza virus, the influenza virus is usually inoculated into the allantoic cavity of a growing chicken egg about 10 days after fertilization. The virus infects only in the allantoic cavity, and the virus accumulates in chorioallantoic fluid. Thereafter, the virus is recovered from the chorioallantoic fluid and concentrated to produce a virus that is a raw material for the vaccine. From this, a virus whole-particle vaccine inactivated with formalin or the like, an HA subunit vaccine produced by decomposing the virus and producing an HA protein fraction, and the like are produced.

However, highly virulent viruses such as subtype H5 and subtype H7, when infected with chicken eggs, the infection spreads to the embryo, and the embryo is lethal early, so a sufficient amount of virus is produced to produce a vaccine. It was extremely difficult to do. In addition, from the viewpoint of safety, a method for producing a vaccine that does not require direct handling of virulent viruses has been desired.

  Therefore, in the present invention, an attenuated vaccine strain used for vaccine production is introduced by introducing an attenuated mutation into the HA gene of H5N1 influenza virus using a reverse genetics technique for producing an infectious virus from a cloned gene. The purpose was to produce.

In order for the influenza virus to infect cells, the HA protein (HA0) must be partially cleaved by trypsin-like proteolytic enzyme and cleaved into HA1 and HA2. When HA1 is adsorbed to the sialic acid receptor on the cell surface, viral particles are taken up into intracellular lysosomes by endocytosis, and membrane fusion occurs under acidic conditions to establish infection. In conventional influenza virus, the C-terminal side of HA protein Gln / Arg-X-Arg is cleaved and cleaved by trypsin-like proteolytic enzyme that exists only in the lungs and upper respiratory tract, and reinfection is established in adjacent cells. Therefore, the site of influenza infection was limited to the airway area (Robert A. Lamb and Robert M. Krug, ' Orthomyxoviridae : The Viruses and Their Replication' in Fields Virology, Third ed., Edited by BN Fields et al. Lippincon-Raven Publishers, Philadelphia, pp.1353-1445, 1996). In contrast, some viruses of the H5 and H7 subtypes, such as the highly pathogenic H5N1 avian influenza virus, have basic amino acids (Arg-X-Lys / Arg-Arg) at the cleavage site of the HA protein. The infection is systemic and fatality is very high because it is cleaved by Furin-like enzymes that exist side by side and are present in a wide variety of cell types.

The present inventors focused on the cleavage site of the HA protein of the H5N1 influenza virus described above, and reduced the number of basic amino acids present side by side, thereby suppressing the effect of proteases on the HA protein on the virus surface. Succeeded in producing an attenuated virus strain with low infectivity. Further, the present attenuated virus strain showed an immunogenicity comparable to that of the H5N1 influenza virus strain used as a base, and was found to be very excellent as a vaccine strain used for vaccine production. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a recombinant influenza virus characterized by having in its genome a base sequence obtained by modifying the protease cleavage site of the influenza virus hemagglutinin gene. Here, in one aspect of the recombinant influenza virus of the present invention, the hemagglutinin type is H5 type. Furthermore, in one aspect of the recombinant influenza virus of the present invention, the amino acid sequence encoded by the base sequence obtained by modifying the protease cleavage site of the hemagglutinin gene is the amino acid sequence described in SEQ ID NO: 1. In one embodiment of the recombinant influenza virus of the present invention, the base sequence obtained by modifying the protease cleavage site of the hemagglutinin gene is the base sequence described in SEQ ID NO: 2. Furthermore, in one aspect of the recombinant influenza virus of the present invention, the underlying influenza virus is H5N1 type. In addition, in one aspect of the recombinant influenza virus of the present invention, the base influenza virus is the A / HK / 156/97 strain.

  Moreover, according to this invention, the influenza vaccine manufactured from the recombinant influenza virus of this invention is provided. Here, in one aspect of the influenza vaccine of the present invention, the influenza vaccine is a live attenuated vaccine, an inactivated virus whole particle vaccine, or a hemagglutinin subunit vaccine.

Moreover, in one aspect | mode of the influenza vaccine of this invention, an influenza vaccine is a DNA vaccine containing DNA which has the DNA which has the base sequence which changed the protease cleavage site of the said hemagglutinin gene. Furthermore, in one aspect of the influenza vaccine of the present invention, the base sequence obtained by modifying the protease cleavage site of the hemagglutinin gene is the base sequence described in SEQ ID NO: 2.

  Furthermore, according to this invention, the pharmaceutical composition containing the influenza vaccine of this invention is provided. Here, in one aspect of the pharmaceutical composition of the present invention, the pharmaceutical composition further comprises one or more influenza vaccines different from the influenza vaccine of the present invention.

  The recombinant influenza virus of the present invention has in its genome a base sequence in which the protease cleavage site of the HA gene has been altered. Therefore, since the HA protein on the surface of the virus particle is less susceptible to proteases and is not cleaved, the recombinant influenza virus becomes an attenuated type with low infectivity. Further, the immunogenicity of the recombinant influenza virus of the present invention retains the antigenicity of the H5N1 influenza virus based on it. Therefore, the recombinant influenza virus of the present invention having low pathogenicity and capable of inducing a protective response against H5N1 influenza with high frequency is highly safe and can be used as an excellent vaccine strain.

  Hereinafter, the recombinant influenza virus of the present invention will be described in detail.

  In producing a recombinant influenza virus, first, viral genomic RNA is purified from a highly toxic influenza virus. For purification, for example, RNeasy Mini Kit (manufactured by Qiagen) is preferably used. Next, the full length of the HA gene is amplified as cDNA by RT-PCR using the above-described viral genomic RNA as a template and a primer specific for the HA gene, and cloning is performed using a vector. Here, as a vector to be used, pUC19 is desirable. Subsequently, a mutation is introduced into the protease cleavage site of the HA gene after cloning. Specifically, in order to reduce a plurality of basic amino acids present side by side, the base sequence of the portion encoding the basic amino acid is deleted, or other amino acids (for example, glutamic acid which is an acidic amino acid, has a hydroxyl group) Mutation is introduced to replace threonine. It is desirable to introduce mutations using the QuickChange Site-Directed Mutagenesis Kit (Stratagene).

Next, by replacing the HA gene of the attenuated avian influenza virus with the HA gene into which the mutation has been introduced by genetic recombination according to the method of Jinami et al. (J. Virol. 74 , 5556-5561, 2000) Obtain recombinant influenza virus. That is, the recombinant influenza virus is produced by transfecting a suitable host cell with a viral RNP (viral RNA nucleoprotein complex) using a conventional method such as the DEAE dextran method, and is propagated, isolated and purified. be able to. Here, examples of host cells include MDBK cells and MDCK cells.

  Since the recombinant influenza virus obtained by the above-mentioned method has low pathogenicity, even if it is inoculated into the fertilized hen's egg allantoic cavity, it will not infect the hen's egg embryo, and it will enter the allantoic cavity without causing the embryo to die. Virus can be propagated. Furthermore, since the recombinant influenza virus has immunogenicity, the virus grown on fertilized chicken eggs can be collected and used as a live attenuated vaccine. Further, inactivation treatment can be performed with formalin or the like, and it can be used as an inactivated vaccine. In addition, it can also be used as an HA subunit vaccine produced from the HA protein fraction that has decomposed the virus. These vaccines can be administered according to conventional methods.

  Furthermore, the mutation-introduced HA gene used for gene recombination in the present invention can be used as a DNA vaccine by linking with an appropriate expression vector. Here, the expression vector is preferably a plasmid vector. Vectors useful in the production of expression vectors include, but are not limited to, constitutive promoters, inducible promoters, tissue-specific promoters, promoters from influenza virus HA proteins, and the like. The DNA vaccine can be administered by selecting and combining appropriate adjuvants such as CpG adjuvant.

  The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.

1. Preparation of genetically engineered virus Using viral genomic RNA purified from highly virulent avian influenza virus A / HK / 156/97 (H5N1) (abbreviated as HK156 in the following text) with RNeasy Mini Kit (Qiagen) The HA gene-specific synthetic DNA primer (forward primer 1: SEQ ID NO: 3 and reverse primer 2: SEQ ID NO: 4) was used to amplify the full-length HA gene (H5 subtype) as a cDNA by RT-PCR, and pUC19 Cloned. Mutations were introduced into the protease cleavage site of the HA gene using QuickChange Site-Directed Mutagenesis Kit (Stratagene) and synthetic DNA primers (forward primer 3: SEQ ID NO: 5 and reverse primer 4: SEQ ID NO: 6). Specifically, deletion of 12 bases (AGAGAGAGAGAAGA) from A at position 1037 to A at position 1048 of the HA gene, A at position 1052 is replaced with G, and A at position 1056 is replaced with C. And G at position 1057 was replaced with C. As a result, the amino acid sequence encoded by the base sequence of the protease cleavage site was changed from the highly toxic type (PQRERRRKKR) to the attenuated type (PQRETR) (FIG. 1). Then, using the method of Jinami et al. (J.Viro. 74 , 5556-5561, 2000), the attenuated avian influenza virus A / duck / HK / 836/80 (H3N1) (abbreviated as HK836 in the following text) The HA gene was replaced with the above-mentioned H5 subtype HA gene into which the attenuating mutation was introduced by the method described above to obtain a genetically modified virus HK / 9-1-1 (H5N1). That is, the RNP purified from the HK836 virus particle, which decomposes and removes only the HA gene, and the RNP of the H5 subtype HA gene reconstituted in vitro are transfected into MDBK cells or MDCK cells to form virus plaques. I let you. The obtained recombinant virus was isolated by plaque isolation using MDCK cells. In addition, the same HK / 9-1-1 was inoculated into the ovary membrane space of fertilized chickens on the 10th day for the production of a pilot vaccine.

2. Verification of pathogenicity (attenuation) in mice and chickens
When 5 x 10 ⁵ pfu virus was nasally inoculated into 4 week-old ddY mice (purchased from Japan SLC Co., Ltd.), all of the highly toxic HK156 vaccinated groups died within 8 days, whereas HK / The 9-1-1 inoculated group showed no symptoms (FIG. 2). Moreover, when compared with LD ₅₀ , it was attenuated by 10 ⁴ or more (Table 1). In addition, 10 ^5.7 EID50 virus was inoculated intracerebrally into 1-day-old chicks (IC), or 10 ^6.7 EID50 virus was intravenously inoculated into 6-week-old chickens (IV). It showed pathogenicity (PI), with the former showing 100% (10/10) and the latter showing 60% (6/10) mortality, whereas the HK / 9-1-1 and HK836 inoculation groups No symptoms were shown (Table 1).

3. Verification of immunogenicity and protection against infection using mice
In order to confirm the effectiveness of the HK / 9-1-1 virus as a live attenuated vaccine or an inactivated vaccine, the highly virulent avian influenza virus A / HK / 483 / In 97 (H5N1) (abbreviated as HK483 in the following text), the immunogenicity and the protective ability against infection were examined using a mouse model animal. In addition, increases in the HI antibody titer and neutralizing antibody titer in the infected mouse serum were confirmed.

Four-week-old ddY mice were inoculated intranasally (immune) with 10 ⁵ pfu of HK / 9-1-1 virus (live vaccine), or 25 μg (0.5 ml) of formalin-fixed inactivated HK / 9-1-1 The virus (inactivated vaccine) was intraperitoneally inoculated (immunized). The control group was inoculated intraperitoneally with 0.5 ml PBS. Three weeks after immunization, 3 mice were killed and serum was obtained. An increase in the serum neutralizing antibody titer against HK156 and HK483 viruses was confirmed (Table 2).

Similarly, 3 weeks after immunization, 1,000 LD ₅₀ of HK156 or HK483 virus was inoculated intranasally (challenge) and observed for 2 weeks. Four days after challenge, 3 mice each were killed and examined for viral growth in the lungs and brain. In the inactivated HK / 9-1-1 immunized group, virus growth was not confirmed in the lung and brain (Table 2). In the live HK / 9-1-1 virus immunized group, slight viral growth was observed in the lung, but it was 10 ^-5 or less compared to the PBS group. HK156 and HK483 in the PBS control group (□) had a fatality rate of 92% and 100%, but the infection protection effect in terms of the lethality rate was 83 to 86% in the inactivated vaccine group (○), live vaccine The group (●) showed 100% protection against any virus (Table 2). Compared with the weight loss of mice, significant infection protection by vaccination could be confirmed (FIG. 3). On the other hand, there seemed to be no significant difference in the protective effect against HK156 and HK483. In addition, an increase in HI antibody titer in infected mouse serum was also confirmed (Table 3).

  The influenza vaccine of the present invention can be used as an effective and safe vaccine against H5N1 influenza, which has recently been reported as a problem.

FIG. 1 is a schematic diagram of a genetic modification site of the recombinant influenza virus of the present invention. Ksp632I represents a restriction enzyme cleavage site, N represents a non-coding region, and T3 represents a T3 promoter site. FIG. 2 is a graph of the survival rate after nasal inoculation of mice with HK / 9-1-1 virus in order to verify pathogenicity. FIG. 3 is a diagram in which infection protection by inoculation with PBS in a live vaccine, inactivated vaccine, or control experiment was confirmed using changes in body weight and survival rate of mice as indices. (A) and (B) show the course after challenge with HK156 strain 3 weeks after immunization, and (C) and (D) show the course after challenge with HK483 strain.

Claims (12)

A recombinant influenza virus characterized in that it has a base sequence modified in the protease cleavage site of the hemagglutinin gene of influenza virus in its genome. The recombinant influenza virus according to claim 1, wherein the hemagglutinin is H5 type. The recombinant influenza virus according to claim 1 or 2, wherein the amino acid sequence encoded by the base sequence obtained by modifying the protease cleavage site of the hemagglutinin gene is the amino acid sequence shown in SEQ ID NO: 1. The recombinant influenza virus according to claim 4, wherein the base sequence obtained by modifying the protease cleavage site of the hemagglutinin gene is the base sequence shown in SEQ ID NO: 2. The recombinant influenza virus according to any one of claims 1 to 4, wherein the influenza virus is H5N1 type. The recombinant influenza virus according to any one of claims 1 to 5, wherein the influenza virus is A / HK / 156/97 strain. An influenza vaccine produced from the recombinant influenza virus according to any one of claims 1 to 6. The influenza vaccine according to claim 7, wherein the influenza vaccine is a live attenuated vaccine, an inactivated virus whole particle vaccine, or a hemagglutinin subunit vaccine. The influenza vaccine according to claim 7, wherein the influenza vaccine is a DNA vaccine comprising a DNA having a base sequence obtained by modifying a protease cleavage site of the hemagglutinin gene. The influenza vaccine according to claim 9, wherein the base sequence obtained by modifying the protease cleavage site of the hemagglutinin gene is the base sequence shown in SEQ ID NO: 2. A pharmaceutical composition comprising the influenza vaccine according to any one of claims 7 to 10. The pharmaceutical product according to claim 9, further comprising one or more influenza vaccines different from the influenza vaccine according to any one of claims 7 to 10.

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